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Vav2 is required for cell spreading.

Marignani PA, Carpenter CL - J. Cell Biol. (2001)

Bottom Line: Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation.A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange.These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA.

ABSTRACT
Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

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Effect of L342R/L343SVav2 on cell spreading. (A) NIH3T3 cells were transfected with vector alone or L342R/L343SVav2. 24 h after transfection they were trypsinized, allowed to recover, and then plated on fibronectin-coated coverslips. 15 or 60 min after plating the cells were fixed and stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. Cells were counted (100 per condition) and scored for spreading: unspread (black bar), partially spread (crossed bar), or fully spread (back-slash bar). Values are expressed as percent of total cells counted and represent the mean ± SEM of four experiments. (B) NIH3T3 cells were transfected with pEGFP and vector only, or L342R/L343SVav2 at a DNA ratio of 1:3 (pEGFP/Vav2 or vector). Cells were trypsinized 24 h after transfection, allowed to recover, and then plated on fibronectin. Cell spreading was monitored by phase contrast microscopy. Photographs were taken at 0, 10, 20, and 40 min (untransfected cell, white arrow; transfected cells, black arrows). These results are typical of 25 cells examined from 3 separate experiments. (C) NIH3T3 cells were transfected with pEGFP and L342R/L343SVav2, trypsinized 24 h later, allowed to recover, and then plated onto fibronectin-coated coverslips. Cells were examined for the presence of filopodia by phase contrast (left, black arrows indicate filopodia) or fixed and stained with rhodamine-phalloidin (middle). Untransfected cells were also plated and stained with rhodamine-phalloidin (right). (D) NIH3T3 cells were trypsinized, allowed to rest, then plated on fibronectin as described in Materials and methods. Cells were harvested at 0, 20, 40, and 60 min after plating, endogenous Vav2 was immunoprecipitated and tyrosine phosphorylation was determined by Western blotting. Total cell lysates (TCL) were also Western blotted for phosphotyrosine. These results are representative of three experiments. Bars, 10 μm.
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fig8: Effect of L342R/L343SVav2 on cell spreading. (A) NIH3T3 cells were transfected with vector alone or L342R/L343SVav2. 24 h after transfection they were trypsinized, allowed to recover, and then plated on fibronectin-coated coverslips. 15 or 60 min after plating the cells were fixed and stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. Cells were counted (100 per condition) and scored for spreading: unspread (black bar), partially spread (crossed bar), or fully spread (back-slash bar). Values are expressed as percent of total cells counted and represent the mean ± SEM of four experiments. (B) NIH3T3 cells were transfected with pEGFP and vector only, or L342R/L343SVav2 at a DNA ratio of 1:3 (pEGFP/Vav2 or vector). Cells were trypsinized 24 h after transfection, allowed to recover, and then plated on fibronectin. Cell spreading was monitored by phase contrast microscopy. Photographs were taken at 0, 10, 20, and 40 min (untransfected cell, white arrow; transfected cells, black arrows). These results are typical of 25 cells examined from 3 separate experiments. (C) NIH3T3 cells were transfected with pEGFP and L342R/L343SVav2, trypsinized 24 h later, allowed to recover, and then plated onto fibronectin-coated coverslips. Cells were examined for the presence of filopodia by phase contrast (left, black arrows indicate filopodia) or fixed and stained with rhodamine-phalloidin (middle). Untransfected cells were also plated and stained with rhodamine-phalloidin (right). (D) NIH3T3 cells were trypsinized, allowed to rest, then plated on fibronectin as described in Materials and methods. Cells were harvested at 0, 20, 40, and 60 min after plating, endogenous Vav2 was immunoprecipitated and tyrosine phosphorylation was determined by Western blotting. Total cell lysates (TCL) were also Western blotted for phosphotyrosine. These results are representative of three experiments. Bars, 10 μm.

Mentions: Spreading of cells on fibronectin involves Cdc42-dependent filopodia formation followed by Rac-dependent lamellipodia and then Rho activation (Clark et al., 1998; Price et al., 1998). To determine whether Vav2 is necessary for cell spreading, NIH3T3 cells were transfected with L342R/L343SVav2 and 24 h later they were trypsinized, allowed to recover, and then plated onto fibronectin. At various time points the cells were fixed and stained for Vav2 expression and for actin. The number of cells that were unspread, partially spread, or fully spread was then determined. L342R/L343SVav2 significantly impaired the spreading of NIH3T3 cells on fibronectin, indicating that Vav2 is necessary for this process (Fig. 8 A).


Vav2 is required for cell spreading.

Marignani PA, Carpenter CL - J. Cell Biol. (2001)

Effect of L342R/L343SVav2 on cell spreading. (A) NIH3T3 cells were transfected with vector alone or L342R/L343SVav2. 24 h after transfection they were trypsinized, allowed to recover, and then plated on fibronectin-coated coverslips. 15 or 60 min after plating the cells were fixed and stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. Cells were counted (100 per condition) and scored for spreading: unspread (black bar), partially spread (crossed bar), or fully spread (back-slash bar). Values are expressed as percent of total cells counted and represent the mean ± SEM of four experiments. (B) NIH3T3 cells were transfected with pEGFP and vector only, or L342R/L343SVav2 at a DNA ratio of 1:3 (pEGFP/Vav2 or vector). Cells were trypsinized 24 h after transfection, allowed to recover, and then plated on fibronectin. Cell spreading was monitored by phase contrast microscopy. Photographs were taken at 0, 10, 20, and 40 min (untransfected cell, white arrow; transfected cells, black arrows). These results are typical of 25 cells examined from 3 separate experiments. (C) NIH3T3 cells were transfected with pEGFP and L342R/L343SVav2, trypsinized 24 h later, allowed to recover, and then plated onto fibronectin-coated coverslips. Cells were examined for the presence of filopodia by phase contrast (left, black arrows indicate filopodia) or fixed and stained with rhodamine-phalloidin (middle). Untransfected cells were also plated and stained with rhodamine-phalloidin (right). (D) NIH3T3 cells were trypsinized, allowed to rest, then plated on fibronectin as described in Materials and methods. Cells were harvested at 0, 20, 40, and 60 min after plating, endogenous Vav2 was immunoprecipitated and tyrosine phosphorylation was determined by Western blotting. Total cell lysates (TCL) were also Western blotted for phosphotyrosine. These results are representative of three experiments. Bars, 10 μm.
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Related In: Results  -  Collection

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fig8: Effect of L342R/L343SVav2 on cell spreading. (A) NIH3T3 cells were transfected with vector alone or L342R/L343SVav2. 24 h after transfection they were trypsinized, allowed to recover, and then plated on fibronectin-coated coverslips. 15 or 60 min after plating the cells were fixed and stained for Vav2 expression with T7 antibody and with rhodamine-phalloidin. Cells were counted (100 per condition) and scored for spreading: unspread (black bar), partially spread (crossed bar), or fully spread (back-slash bar). Values are expressed as percent of total cells counted and represent the mean ± SEM of four experiments. (B) NIH3T3 cells were transfected with pEGFP and vector only, or L342R/L343SVav2 at a DNA ratio of 1:3 (pEGFP/Vav2 or vector). Cells were trypsinized 24 h after transfection, allowed to recover, and then plated on fibronectin. Cell spreading was monitored by phase contrast microscopy. Photographs were taken at 0, 10, 20, and 40 min (untransfected cell, white arrow; transfected cells, black arrows). These results are typical of 25 cells examined from 3 separate experiments. (C) NIH3T3 cells were transfected with pEGFP and L342R/L343SVav2, trypsinized 24 h later, allowed to recover, and then plated onto fibronectin-coated coverslips. Cells were examined for the presence of filopodia by phase contrast (left, black arrows indicate filopodia) or fixed and stained with rhodamine-phalloidin (middle). Untransfected cells were also plated and stained with rhodamine-phalloidin (right). (D) NIH3T3 cells were trypsinized, allowed to rest, then plated on fibronectin as described in Materials and methods. Cells were harvested at 0, 20, 40, and 60 min after plating, endogenous Vav2 was immunoprecipitated and tyrosine phosphorylation was determined by Western blotting. Total cell lysates (TCL) were also Western blotted for phosphotyrosine. These results are representative of three experiments. Bars, 10 μm.
Mentions: Spreading of cells on fibronectin involves Cdc42-dependent filopodia formation followed by Rac-dependent lamellipodia and then Rho activation (Clark et al., 1998; Price et al., 1998). To determine whether Vav2 is necessary for cell spreading, NIH3T3 cells were transfected with L342R/L343SVav2 and 24 h later they were trypsinized, allowed to recover, and then plated onto fibronectin. At various time points the cells were fixed and stained for Vav2 expression and for actin. The number of cells that were unspread, partially spread, or fully spread was then determined. L342R/L343SVav2 significantly impaired the spreading of NIH3T3 cells on fibronectin, indicating that Vav2 is necessary for this process (Fig. 8 A).

Bottom Line: Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation.A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange.These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA.

ABSTRACT
Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

Show MeSH
Related in: MedlinePlus